AU706613B2 - Supplement for baby infant formula and a method of delivering that supplement - Google Patents

Description

WO 96/26647 PCT/AU96/00103 1 SUPPLEMENT FOR BABY INFANT FORMULA AND A METHOD OF DELIVERING

THAT

SUPPLEMENT

The present invention relates to a supplement for baby infant formula and a method of delivering that supplement.

In particular, the invention relates to a stable delivery system of a supplement of long chain polyunsaturated fatty acids docosahexaenoic acid 22:603 (DHA), eicosapentaenoic acid 20:5o3 (EPA), gamma linolenic acid 18:30o6 (GLA) and arachidonic acid 20:4w6 (AA) from tuna oil in combination with evening primrose oil to infant milk formula so that the formula approximates breast milk. These long chain fatty acids are notoriously unstable. When added to baby infant formula this supplement improves neurological visual evoked responses in preterm and term babies. When given in appropriate doses, it maintains normal levels of AA and higher levels of DHA and GLA which other fish oils do not.

The invention also relates to a method of treatment using the supplement of this invention.

Background Art It has been known for years the adequate supply of two essential fatty acids, alphalinolenic acid 18:303 (ALA), and linoleic acid 18:20o6 (LA) are important in infant formula and all infant formulas contain vegetable oils that are rich in these essential fatty acids.

New research into the roles and effects of dietary fat supply in infancy show that the way babies develop is profoundly affected by the fat supply. Of particular interest are the results of research into the roles of two long chain polyunsaturated fatty acids known as docosahexaenoic acid (DHA) and arachidonic acid (AA) which indicate new concepts in the dietary requirement for infants.

Studies comparing breast and formula feeding indicate that formula fed infants have less DHA and AA in their cells relative to breast fed infants. Studies which tried to increase ALA (precursor to DHA) to formula fed infants demonstrated that it was only possible to increase DHA levels to 80% of those breast fed infants.

These results indicated that it was not possible to correct the balance of DHA and AA by altering the vegetable oil content of infant formula. -In other words, DHA and AA are required to be present in the formula along with an oil containing GLA.

Present compositions of fish oil have high EPA and DHA with a total Omega-3 of (18% EPA, 12% DHA). When fish oil of this type is supplemented to infant formula there is an increase in DHA in the infants, but a lowering of AA. The AA in fish oil supplemented to infants was reduced to below standard formula levels due to high levels of EPA.

These long chain polyunsaturated fatty acids are known to be unstable and the research by major infant formula companies is to make a stable DHA and AA powder SUBSTITUTE SHEET (RULE 26) which is combined in the infant formula. Shelf life is about six months at temperatures below 25 0 C which does not make it a realistic product placed on the pharmacy shelf with an expected life of 1-2 years. Unless the stability can be improved these powders will have limited success in the infant formula marketplace.

It has been found that fish have various compositions of polyunsaturated fatty acids depending upon geographical position and available food chain. Reference is made to Table 1 which sets out the polyunsaturated acids found in various species of fish.

Table 1 Polyunsaturated Fatty Acids in Fishl, 2 Fish (Total Fat) 18:3o6 20:4o6 20:50c3 22:6o)3 Tuna (Albacore) 0.097 0.110 0.37 1.10 Anchovy (European) (4.84) 0.108 0.008 0.596 1.009 Herring (Atlantic) 0.142 0.066 0.0774 0.0940 Mackerel (Atlantic) (13.9) 0.425 0.200 1.1620 2.465 Salmon (Chinook) (10.4) 0.115 0.167 0.857 0.617 Tuna (Bluefin) 0.080 0.065 0.423 1.327 Halibut (Pacific) 0.036 0.160 0.083 0.338 Flounder 0.006 0.044 0.114 0.110 Cod (Atlantic) (0.75) 0.009 0.024 0.114 0.191 Haddock 0.071 0.173 0.443 0.943 6 6 Sr.

6 6 6 Expressed as grams per 100 grams of food 2 Data from Kinsella, J.E. Seafoods and Fish Oils in Human Health and Disease. Marcel Dekker Inc. New York and Basel Disclosure of the Invention According to a first embodiment of this invention there is provided a supplement for addition to an infant milk formula comprising a blend of docosahexaenoic acid 22:60o3 (DHA), eicosapentaenoic acid 20:5(o3 (EPA), gamma linolenic acid 18:30o6 (GLA) and arachidonic acid 20:4o)6 (AA) at a level sufficient to approximate the level found in natural breast milk encapsulated in soft gelatin. Preferably, the supplement is effective in increasing DHA in infants being fed the formula and/or is effective in maintaining high levels of AA in these infants and/or is effective in increasing GLA in these infants.

The specification describes a supplement for addition to an infant milk formula comprising a blend of essential fatty acids at a level sufficient to approximate the level found in natural breast milk encapsulated in a suitable material. Preferably, the capsule is soft gelatin and is provided with a teat which for example is able to be snipped, thereby allowing the contents of the capsule to be discharged into standard milk formulation whenever required. This thus allows easy administration of the blend and an ability to control and dispense the blend into the formulation.

N:\LIBVV]0894:SSD According to a second embodiment of this invention there is provided a method for improving the visual evoked response in infants, which method comprises administering to said infants an effective amount of supplement for addition to an infant milk formula comprising a blend of docosahexaenoic acid 22:6c3 (DHA), eicosapentaenoic acid 20:5m 3 (EPA), gamma linolenic acid 18:3m6 (GLA) and arachidonic acid 20:4c6 (AA) at a level sufficient to approximate the level found in natural breast milk.

4 WO 96/26647 PCT/AU96/00103 3 comprising a blend of essential fatty acids at a level sufficient to approximate the level found in natural breast milk.

Typically, the DHA, EPA and AA are extracted from phytoplankton, zooplankton, bacterial polyunsaturated fatty acid producers, and fish. The blend preferably is prepared from oil extracted from fish and is more preferably extracted from tuna fish and mullet ie.

fish of the Scombridae and Mugilidae families. Species of tuna fish which are suitable are albacore, bluefin, yellowfin and western pacific skipjack. Tuna oil has a preferred ratio of DHA:EPA. Although tuna oil is preferably used as a source of DHA, EPA, and AA, any fish which provides suitable levels of these long chain unstable fatty acids will suffice. A further source of preferred blends of fatty acids may be extracted from algae.

The fish oil preferably contains high levels of DHA of between about 25-30% of total fat and a DHA:EPA ratio of about 4.0-7.0:1 and AA of about 1.7-2.5% of total fat together with a evening primrose oil in a blend of about 80% fish oil to about evening primrose oil. The evening primrose oil contains a GLA of 10% or more.

As mentioned, the blend also preferably contains evening primrose oil. This is suitably prepared by cold pressing and filtering or from solvent extracts containing essential fatty acids or their metabolites or using supercritical extraction techniques.

Suitable solvents when using the solvent extraction method are for example hexane or isopropanol.

The blend also suitably contains an emulsifer or blends of emulsifiers. For example, a typical emulsifier is lecithin.

The blend may also contain antioxidants such as mixed natural tocopherols.

If desired, the blend may also contain trace elements such as selenium; coenzymes such as coenzyme Q10; minerals such as calcium; other essential fatty acids; and other herbal extracts.

If desired, the blend may also contain vitamins. For example, it may be desirable to add vitamins in geographical areas where such a vitamin is below recommended levels or deficient. Particularly, fat soluble vitamins such as vitamin E may be added.

Best Modes and Other Modes for Carrying Out the Invention The procedure of blending is carried out under pharmaceutical good manufacturing practise as defined by the Australian Therapeutic Goods Administration.

The oils are passed by quality assurance as meeting the specifications for raw material as defined.

Four parts of refined, high DHA, oil (sourced from a marine species), measured by weight, is blended under nitrogen with one part of refined, high GLA, evening primrose oil, measured by weight. For example, four kilograms of high DHA oil would be blended with one kilogram of high GLA oil. This process is conducted in an atmosphere which is significantly reduced in oxygen by saturating the blending environment with food grade nitrogen.

SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 4 The blended oil is mixed slowly is a stainless steel vessel until full mixing has occurred. Care must be taken not to aerate the blended oils.

The mixing vessel is capped under food grade nitrogen nad taken to the encapsulation room. The blended oil is then encapsulated into 500gm soft gelatine capsules.

The present invention will now be described with reference to the following examples which should not be construed as limiting on the scope thereof.

Example 1 Production and Processing of Tuna Oil (fish oil) The raw material for the production of fish or tuna oil fall into two classes. The first category of raw materials is the pelagic-type fish specifically pursued for reduction to meal and oil. The second category is from facilities that produce edible fish such as salmon or tuna.

The universal process used to produce fish or tuna oil is the wet rendering process.

Major steps in the process are cooking, pressing, drying and separating.

Cooking coagulates the fish protein so that the liquid and solids can be mechanically separated. The oil is then separated from the pressliquid by centrifugation.

The crude tuna oil contains impurities which reduce the acceptability because of the flavours and odours and also reduce the stability and shelf life of the oil. To produce a safe, edible product the crude oil has to undergo certain processing steps which include degumming, neutralisation, bleaching, winterisation and deodorisation.

The degumming step removes hydrated gums, phosphorous and trace metals. The subsequent alkali treatment neutralises the free fatty acids and removes the hydrated gums with the soapstock. Bleaching is used to improve the colour, flavour and oxidative stability of the oil and also impurities, such as traces of soap.

Winterisation removes the higher-melting glycerides from the oil and deodorisation is the last major processing step to remove and destroy peroxides, secondary and tertiary oxidation products and other volatile product which affect the stability of the oil and the organoleptic acceptability as an edible oil.

Example 2 Production Method for Evening Primrose Oil There are two preferred methods for the production of evening primrose oil: i. Mechanical extraction method "Cold Pressing"; and ii. Solvent extraction method.

Prior to production in either method, the seed is collected, cleaned and graded according to gamma linolenic content.

i. Mechanical extraction method "Cold Pressing" The seed is mechanically pressed using a screw press at a maximum temperature of about 50C. The crude oil contains impurities which reduce the consumer acceptability SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 because of flavours and odours and can reduce the shelf life of the oil. To produce a safe, edible oil product the oil has to undergo certain processing steps which include degumming, neutralisation, bleaching, winterisation and deodorisation.

The degumming step removes hydrated gums, phosphorous and trace metals. The subsequent alkali treatment neutralises the free fatty acids and removes the hydrated gums with the soapstock. Bleaching is used to improve the colour, flavour and oxidative stability of the oil and also impurities, such as traces of soap.

Winterisation removes the higher-melting glycerides from the oil and deodorisation is the last major processing step to remove and destroy peroxides, secondary and tertiary oxidation products and other volatile product which affect the stability of the oil and the organoleptic acceptability as an edible oil.

ii. Solvent extraction method The evening primrose seeds are rolled, or "prepressed", to crack the seed producing a cake, with final extraction of the oil achieved by solvent extraction. The solvent, usually hexane, leaches the oil out of the cake. The leached oil and solvent form a mixture which is referred to as "Miscella". It is essential to have enough clean solvent to keep miscella low enough in concentration to dissolve and displace more oil at each stage of extraction. The process occurs under gentle stirring, in an extractor, to ensure good contact and penetration, such that solvent contacts oil and is able to' flow out of the particles with the oil.

As the cake is passed through the extractor it is washed with lower and lower concentrations of oil in hexane and finally washed with clear hexane prior to leaving the extractor.

The miscella from the extractor contains about 22-30% oil and the rest is solvent.

The basic process is to heat this mixture under a vacuum and evaporate out the solvent, leaving the oil behind. The oil is then steam stripped in a counterflow process and goes into storage as crude oil. The solvent vapours are condensed, separated from water, heated and sent back to the extractor. This procedure may be repeated to reduce the solvent content in the oil to below 1 ppm or lower. Residual solvent is extracted at a maximum temperature of about 50 to about 60 0

C.

The crude oil is then refined in the same manner as evening primrose oil detailed in above.

Example 3 Blending of Oils Tuna oil and Evening Primrose oil were mixed together at precise ratios (w/w) under nitrogen.

Example 4 Measurement of Visual Evoked Response in Infants SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 6 Visual evoked response, expressed as visual evoked potential or VEP, is measured according to Sokol, S. Vision Research 18, 33-39 as follows. Infants sat with their mothers 1 metre away from a monitor that presented high-contrast black-and-white checkerboard-pattern stimuli. A constant reversal rate of 2/s was used to elicit responses to checks with squares subtending visual angles of 7, 17, 34, 69 and 137 min of arc.

Responses were obtained from an active electrode placed 3cm above the inion, a reference electrode in a midfrontal position and an inactive electrode of the forehead. Recording occurred when infants were alert and their gaze directed at the monitor. A bell was used to help maintain infants' attention. Three records were taken at each check size to ensure reproducibility.

Peak-to-peak amplitude was determined for each check size. The amplitude of the evoked potential diminishes as check size decreases. Extrapolation of a regression line fitting VEP amplitude to log (check size) through 0-pV amplitude gives the smallest check size that, if tested, would render a positive wave. This point is defined as the logMAR or VEP acuity.

VEP actuity of infant formula fed infants and formula fed infants supplemented with the recommended dose of the blended oil containing the long chain polyunsaturated fatty acids: Formula Fed Formula Fed and Supplemented with AA, DHA,

GLA

VEP Acuity 0.74±0.27 0.35±0.29* p<0.01 Example Plasma Levels of Fatty Acids of DHA, EPA and AA (mg/L) in infants on Infant Formula Blood samples 150 to 200 ul whole blood was obtained from a heel prick and transferred to tubes containing ethylenediamine tetracetic acid. After removal of plasma, erythrocytes were washed three times with cold isotonic saline solution and the lipids were extracted immediately.

Lipid extraction Total lipids were extracted from plasma and infant formula with chloroform/methanol (2:1 vol/vol) and from erythrocyte membranes with chloroform isopropanol (2:1 vol/vol).

Fatty Acid methylation Plasma, erythrocyte, and infant formula lipid extracts were evaporated to dryness under nitrogen. The samples were methylated in 1% H2S0 4 in methanol at 70 0 C for 3 hours. After cooling, the resulting methyl esters were extracted into n-heptane and transferred to vials containing anhydrous Na-SO 4 as the dehydrating agent.

Gas chromotographic analysis of fatty acid methyl esters Fatty acid methyl esters were separated and quantified with a Hewlett-Packard 5880 gas chromatograph equipped with a 50cm capillary column (0.56 mm inside diameter) coated with SP2340.

SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 7 The injector temperature was set at 250°C and the detector (flame ionization detector) temperature at 300 0 C. The initial over temperature was 120°C and was programmed to rise to 200 0 C at 5°C/min. Helium was used as the carrier gas at a velocity of Fatty acid methyl esters were identified based on the retention time to authentic lipid standards obtained from Nu-Check, Inc.

and Infant Formula Supplemented with High DHA Tuna nil IR( )HA EPA

AA

1 2 3 4 0 1 2 3 4 0 1 -23 4 .7 0.6 0.9 0.7 0.6 0.6 0.7 0.6 0.4 0.3 3.6 4.0 3.2 3.0 2.6 .8 1.9 1.6 1.7 1.6 0.6 0.6 0.5 0.4 0.5 9.7 9.2 8.6 8.8 8.1 (o A n I Q =M -1 3.0 u.7 2.5 0.7 1.8 2.J 2.9 2.8 3.5 2.6 3.0 2.6 3.2 1.7 1.6 1.9 10.0 9.8 10.2 10.1 10.5 Weight of Infants at study (g) Age (day) Plasma Phospholipids A 1313 ±135 B 1167 ±135 A 23±5 B 22 Example 6 MMW== Typical Fatty Acid Analysis by Capillary Gas Chromatography of Tuna Fish Oil (Clover Corp Tuna B) 8. U.uy 10:0 0.02 irans 16:1 Trans 18:1(o9 1:2(O0 U.79 I l- AtA \^Cr i- KKBS g B BS 0.10 16:1(o9 0.27 18:3o6 0.26 18:43o3 0.49 12:0 12:0 I I-s Trans 18:l)7 10:1o/ 20:2(o6 20:5(o3 3.89 14 It:0 15:0 1.17 irans 18:2 Total Trans 20:30)6 0.15 22:5073 110.81 0.56 18: 1o9 14.58 20:406 2.04 22:6(3 25.74 dma 16 16:0 17:0 .0 T-029 1 l.t 10.1) 22:30)6 Total m3 31 18 1Total3 31 18 2.

20U: 11 1 22:3056 22Iwl .20:1o 9 Tota l.6 dma 18:0 0.16 20:3 o9 18:0 7.08 22:1ol1 0.27 20:0 0.39 22:1m9 0.02 24:0 U U.-U 24:1a)9 1 (10 II. II 41 Total Monos 22.99 Total 39.0 Total o9 15.30 Sets Total (o7 6.84 The above analysis was carried out using method Ce lb-89 of the American Oil Chemists Society.

Example 7 Typical Fatty Acid Analysis by Capillary Gas Chromatography of Evening Primrose Oil Fatty Acid wt% Palmitic 16:0 5.9 Stearic 18:0 Arachidic 20:0 0.3 Beheric 22:0 0.1 Oleic 18:1o9 5.7 18:1co7 0.6 SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 The above analysis was carried out using method Ce lb-89 of the American Oil Chemists Society.

Example 8 Tvoical Sthaility of Rlister P-ac I r 0 6 Months 12 Months Acid Value mg KOH/g <0.10 <0.10 <0.10 Peroxide Value mg equiv/Kg <5.0 <5.0 Anisidine Value <20.0 <20.0 <20.0 The acid value was determined using method Cd 3d-63 of the American Oil Chemists Society. The peroxide value was determined using method Cd 8b-90 of the American Oil Chemists Society. The anisidine valuewas determined using method BS684 2.24:1989.

The according Example 9 following table sets out an example of the contents of a typical capsule to this invention together with the constitution of the shell of such a capsule.

1. Softgel Fill Ingredient(s): Evening Primrose Oil (equiv. Linoleic Acid (equiv. gamma-Linolenic Acid Per Softgel 100mg Fish Oil Natural (Tuna) 400mg (equiv. Omega-3 Triglycerides 132mg) (equiv. Docosahexaenoic Acid 108mg) (equiv. Eicosapentaenoic Acid 17mg) (equiv. Arachidonic Acid 8.4mg) 2. Softgel Shell Standardised Composition: mg per softgel) mg Gelatin 167mg Glycerol 94.3 Water purified 22.7 Uniformity of mass complies to Therapeutic Goods Order TGO 36 (Clauses 5.1(b)) Softgel Capsule Size/Shape: 9 minim tube Weights Fill 500 Shell (approx.): 284 Example Addition of Capsule Contents to Milk Supplement As a general rule, one drop of the supplement, an instance of which appears in Example 9, is added to 30mL of milk formula.

SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 9 Example 11 Tuna Fish Oil: Raw Material Specification Acid Value mgKOH/g Peroxide Value Meq02/kg Anisidine Value Gardner Specified Fatty Acids Triglycerides Docosahexaenoic Acid Arachidonic Acid Maximum Limit Eicosapentaenoic Acid Antioxidants Mixed natural tocophenols Pesticides Total PCB's mg/kg Total DDT's mg/kg Heavy Metals Arsenic mg/kg Cadmium mg/kg Lead mg/kg Mercury mg/kg Copper mg/kg Iron mg/kg Microbiology Total plate count c.f.u./g Yeast and mould count c.f.u./g Enterobacteriacea c.f.u./g E. Coli/g Salmonella/1Og Example 12 Evening Primrose Oil: Raw Material Specification Acid Value mgKOH/g Peroxide Value Meq02/kg Anisidine Value Gardner Fatty Acid Profile Triglycerides Linolenic Acid Gamma linolenic acid Antioxidants Mixed natural tocophenols Pesticides Total PCB's mg/kg Total DDT's mg/kg Heavy Metals Arsenic mg/kg Cadmium mg/kg Lead mg/kg Mercury mg/kg Max. 2 Max. Max. 7 Min 95% Triglyceride 220.0mg/g-255.2mg/g 1 7 Max. 7 9 .2mg/g 2mg/g <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 100

ND

ND

Max. 1 Max. Max. 7 Min 95% Triglyceride 7 15mg/g-831mg/g 88mg/g-102mg/g 2mg/g <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/AU96/00103 Copper mg/kg Iron mg/kg Microbiology Total plate count c.f.u./g Yeast and mould count c.f.u./g Enterobacteriacea c.f.u./g E. Coli/g Salmonella/1Og Example 13 Tuna Fish: Evening Primrose Oil (4:1 W/W Blend) Analysis Acid Value mgKOH/g Peroxide Value Meq02/kg Anisidine Value Microbiology Total plate count c.f.u./g Yeast and mould count c.f.u./g Enterobacteriacea c.f.u./g E. Coli/g Salmonella/i Og <0.1 <0.1 100

ND

ND

Release Specification Result Max.2 Max.8 100

ND

ND

Range 92.5%-107.5% (71.5mg-83.1mg) 8 .8mg-10.2mg) (87.0Omg-102.1mg) 7.0mg-8.2mg) Ingredients *Based on min Triglycerides Evening Primrose Oil equiv. Lineolic Acid equiv. Gamma Linolenic Acid Fish Oil Tuna equiv. Docosahexaenoic Acid equiv. Arachidonic Acid Maximum Limit Eicosapentaenoic Acid Per Capsule 100mg 77.3mg 9.5mg 400mg 95.0mg 7.6mg 3.17mg Example 14 Tuna Fish: Evening Primrose Oil Analysis Acid Value mgKOH/g Peroxide Value Meq02/k Anisidine Value Microbiology Total plate count c.f.u./g Yeast and mould count c Enterobacteriacea c.f.u./ E. Coli/g Salmonella/1 Og (4:1 W/W Blend) Shelf Life Specification Result Max.2 g <100

ND

ND

Ingredients *Based on min Triglycerides Evening Primrose Oil Per Capsule 100mg Range 90%-110% SUBSTITUTE SHEET (RULE 26) WO 96/26647 PCT/A1J96/oo 103 equiv. Lineolic Acid equiv. Gamma Linolenic Acid Fish Oil Tuna equiv. Docosahexaenoic Acid equiv. Arachidonic Acid 11 7 7.3mg 9.5mg 400mg 95.0mg 7.6mg 6 9 .6mg-85 .0mg) 8 6 mg-1O.4mg) 8 5 6 mg-104.5mg) 6 .8mg-8.4mg) SUBSTITUTE SKEET (P~ULE 26) Stability Data 4:1 (wlw) Blend of Tuna Fish Oil and Evening Primrose Oil encapsulated in soft gelatine capsules, blisterpacked in 130-140 micron rigid0 PVC Film, unplasticised and packed into a cardboard box. Cardboard outer box. Capsule Content: Two batches of the product (Total of 30 boxes per batch comprising 2 x 15 capsule blisterpacks of softgel capsules per cardboard box) were 'Placed in ovens at 25*C and 35*C.

Tested at time of Softgel Encapsulation (Zero Time and at fixed intervals thereafter) Table 12 Zero time One month Two months Three months Six Months Batch 7728E 7728EB 7728E 7728EB 7728E 7728EB 7728E 7 /28EB 772SE 7728EB 7 128E 7728EB 7728E 7728EB 112613 7728EB 7728E 772SEB A A A A A A A A

A

No. 25*C 5C 35C 5 2525C 2 5 0 35*C 35 0 C 25 0 25 *C 3.5,C 35 0 C 25 0 C 25 C 35 0 C 350C FFA 0.2 0.24 0.1 0.1 0.1 0.1 0.2 02 0.2 0.1I 0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 P 5.2 5.0 5. 52 5.3 5.7 5.3 5.7 5.3 5.6 5.8 5.9 5.8 5.9 6.3 3.0 5.5 3.0 C3LAo" 16.63 16.90 16.40 16.71 16.90 16.26 16.94 16.73 16.19 16.05 16.38 16.43 16.42 16.19 16.21 16.17 16. 31 Dama COGLA% 2.27 2.19 2.34 2.19 2.42 2.20 2.47 2.27 2.10 2.10 2.15 2.16 2.14 2.12 2.0 .9 218 n AA%' 1.62 1.59 1.61 1.61 1.66 1.62 1.64 1.61 1.62 1.63 1.1 .6 162 .0 157 .6 156 Tas C/ DHA% 23.28 22.62 23.29 22.90 23.79 23.78 23.-78 2312 236 2.8 2.2 2.0 235 2.1 2.2 221 2.9 M15.80 16.06 15.58 15.88 16.06 15.45 16.09 15.89 15..38 15.23 15.56 15.61 15.60 153 15.40 153 9% 2.16 2.08 2.13 2.085 2.30 2.09 2.35 2.16 2.00 20 2.04 2.05 2.03 2.1 2.09 2.8 2.07 deltha tocopherol 1332 1325 Ansdn vle(V) 496.0 WO 96/26647 PCT/AU96/00103 13 Industrial Applicability It should be clear that the formulations and methods used in this invention will find wide use in the infant feeding area.

The foregoing describes only some embodiments of the present invention and modifications obvious to those skilled in the art can be made thereto without departing from the scope of the invention.

SUBSTITUTE SHEET (RULE 26)

Claims (13)

1. A supplement for addition to an infant milk formula comprising a blend of docosahexaenoic acid 22:603 (DHA), eicosapentaenoic acid 20:5Co3 (EPA), gamma linolenic acid 18:3co6 (GLA) and arachidonic acid 20:4co6 (AA) at a level sufficient to approximate the level found in natural breast milk encapsulated in soft gelatin.

2. The supplement according to claim 1 wherein the blend of essential fatty acids is effective in increasing docosahexaenoic acid 22:6c03 in infants being fed the formula and/or is effective in maintaining high levels of arachidonic acid 20:40)6 in these infants and/or is effective in increasing gamma linolenic acid 18:3co6 in these infants.

3. The supplement according to claim 1 or claim 2 wherein the blend is prepared from oil extracted from fish, phytoplankton, zooplankton, bacterial polyunsaturated fatty acid producers, transgenic fish or algae.

4. The supplement according to claim 3 wherein the fish is from the Scombridae or Mugilidae family.

5. The supplement according to claim 4 wherein the fish is tuna.

6. The supplement according to claim 5 wherein the tuna is albacore, bluefin, yellowfin or Western Pacific skipjack.

7. The supplement according to any one of claims 1 to 6 wherein docosahexaenoic acid 22:6co3 is between about 25 to 30% of the total fat and the docosahexaenoic acid 22:603: eicosapentaenoic acid 20:5o03 ratio is 4.0 to 7.0:1 and the arachidonic acid 20:4o06 is between about 1.7 to about 2.5% of the total fat; together with evening primrose oil in a ratio of about 80% fish oil to about 20% evening primrose oil thereby producing a gamma linolenic acid 18:3co6 of about

8. The supplement according claim 7 wherein the evening primrose oil is 25 prepared by cold pressing and filtering or from solvent extracts containing essential fatty acids or their metabolites.

9. The supplement according to any one of claims 1 to 8 further comprising an emulsifier and/or antioxidant and/or a trace element and/or a coenzyme and/or vitamins and/or minerals and/or fatty acids and/or herbal extracts. 30

10. The supplement according to claim 9 wherein the emulsifier is lecithin; the antioxidant is a mixture of natural tocopherols; the trace element is selenium; the coenzyme is coenzyme Q10; the mineral is calcium and the vitamin is vitamin E.

11. The supplement according to claim 1 wherein the gelatin is provided with a teat.

12. A method for improving the visual evoked response in infants, which method comprises administering to said infants an effective amount of supplement for addition to an infant milk formula comprising a blend of docosahexaenoic acid 22:60)3 (DHA), eicosapentaenoic acid 20:503 (EPA), gamma linolenic acid 18:30)6 (GLA) and arachidonic acid 20:4o06 (AA) at a level sufficient to approximate the level found in q RA^, 40 natural breast milk. N:\LIBVV]0894:SSD

13. A supplement for addition to an infant milk formula, substantially as hereinbefore described with reference to any one of Examples 9, 10 or 14 or Table 12. Dated 19 April, 1999 Clover Corporation Pty Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON a a a a. a a S a a. 9~ ~a. a. a. a 7 N:\LBVV]O894:SSD